- Email: [email protected]
RT-PCR evaluation of viral contamination in five shellfish beds over a 21-month period
~
Pergamon
War. ScL
PIT: S0273-1223(98)00798-7
T~ch .
Vol. 38. No. 12. pp. 4S-S0. 1998. e 1998IAWQ Published by Elsevier Science lid
Printed in GreatBritain. All rightsreserved 0273-1223/98 $ 19-00 + 0 '00
RT-PCR EVALUAnON OF VIRAL CONTAMINATION IN FIVE SHELLFISH BEDS OVER A 21-MONTH PERIOD F. Le Guyader*, L. Miossec*, L. Haugarreau*. E. Dubois*, H. Kopecka** and M. Pommepuy* • Laboratoire de Microbiologie, IFREMER. BP 21105.44311 Nantescedex03. France •• Unitede YirologieMoliculaire. lnstitut Pasteur. 28 rue du Dr Raux; 75/24 Pariscedex /5. France
ABSTRACf Five shellfish beds were sampled for 21 months and evaluated for microbial contamination . Viral extraction was performed on dissected tissues and the clinically most important enteric viruses (hepatitis A virus. small round structured virus. rotavirus and enterovirus) were searched for by RT·PCR and hybridization. Among the 104 samples analysed. 66% were contaminated by at least one virus and 34% were negative for any virus. The two sites regularly contaminated by faecal coliforms had the highest percentage of viral contamination and HAV was detected only in these sites. However. sampling sites meeting the criteria for commercialisation showed occasional viral contamination and viruses were detected in samples with no faecal coliform contamination . e 1998 fAWQ Published by Elsevier Science Ltd. All rights reserved
KEYWORDS Enterovirus; faecal colifomns; hepatitis A virus; rotavirus; shellfish; small round structured virus. INTRODUCfION Shellfish are known to concentrate microorganisms such as bacteria or viruses in association with human waste or bacterial pathogens indigenous to coastal marine environments (Rippey, 1994). It is known that standards based on faecal coliforms (FC) and established to protect shellfish consumers are not correlated with the presence of viruses. In recent years, different methods have been developed to detect viral contamination in shellfish (Atmar et al., 1995; Lees et al., 1995; Cromeans et al., 1997). The latest developments in shellfish viral bio-accumulation and current knowledge about viruses at the molecular level have led to the elaboration of a specific, sensitive method which was evaluated in a COllaborative study with artificially contaminated shellfish (Atmar et al.• 1995, 1996), and used to analyse shellfish implicated in an outbreak (Le Guyader et 01.• 1996b). This paper describes the use of this method in assessing the viral Contamination of five different shellfish beds over a 21-month period. The clinically most important viruses likely to be accumulated by shellfish were evaluated by RT-PCR and hybridization: hepatitis A virus (HAV), small round structured virus (SRSV), rotaviruses (RV) and enteroviruses (BV).
45
46
F. LE GUYADER et al.
MATERIALS AND METHODS
Shellfish sampling andJ'rocessing - oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis) were collected monthly from August 1995 to April 1997 at five different sites in southern France known to have different levels of bacterial contamination. Two mussel beds (A-B) were frequently contaminated by faecal coliforms and Salmonella and three oyster beds (C-E) were occasionally contaminated by FC. A total of 42 mussel samples and 63 oyster samples were collected. Shellfish received by the laboratory two days after collection were shucked and the stomach and digestive diverticula removed by dissection. Dissected tissues were frozen (-20°C) in aliquots of 1.5g until processing. For analysis, the tissues were thawed on ice and processed as described previously (Atmar et al., 1995). Briefly, the stomach and digestive diverticula were homogenized with a Potter-Elvehjem homogenizer (Bioblock scientific, Illkirch) in 2mL of phosphatebuffered saline solution (PBS, pH 7.4). The potter was rinsed with an additional 3mL of PBS which was then added to the homogenate. After addition of one volume of chloroform-butanol (I: I v/v), the mix was vortexed for Imin and 90J.LL of Cat-Floc (Calgon Corp, Elwood, Pa) were added. After rocking for 5min and settling for 15min, the mixture was centrifuged at lO,oooxg for 20min, rocked and centrifuged. The aqueous phase was then added to 3mL of polyethylene glycol 6,000 (Sigma, 24% (w/v)-sodium chloride (1.2M) solution). After rocking for lh at 4°C, the solution was centrifuged for 30min at lO,oooxg. The pellet was then suspended in 3mL of sterile water and digested with proteinase K (Sigma) for 30min at 56°C. The nucleic acids were extracted with an equal volume of phenol-chloroform-isoamyl alcohol (25:24: I) (Sigma) and precipitated with ethanol. After the resulting pellet was suspended in water, cetyltrimethylarnrnonium bromide (CTAB) (1.4% w/v) and sodium chloride (Sigma, O.IIM) were added. The mixture was incubated for 15min at room temperature before being centrifuged for 30min at 15,oooxg at 25°C. The resulting pellet was suspended in IM sodium chloride and precipitated in ethanol before suspension in 50J.LL of water prior to reverse transcription.
Intemal control - to evaluate the presence of inhibitory compounds in the enzymatic reactions, internal controls (IC) were used in RT-PCR. A single-strand RNA IC (ssRNA IC) was constructed from the enterovirus genome (Le Guyader et al., 1997) and a double strand RNA IC (dsRNA IC) was constructed from the rotavirus genome (Dubois et al., submitted). Both IC were quantitated by RT-PCR and by spectroscopy at a wavelength of 260nm before use in RT-PCR. Primers - for enterovirus detection two sets of primers both located in the 5' untranslated region were used. One set, KI (162-182) and K2 (577-596) (Kopecka et al., 1993), amplified both endogenous RNA and the ssRNA IC, whereas the second set, EI (446-463) and E2 (623-642) (Chapman et al., 1990; Rotbart, 1990), amplified only endogenous RNA. A specific oligonucleotide probe (533-559) (Chapman et al., 1990) was used to hybridize this fragment. For hepatitis A virus, primers HI (2389-2413) and H2 (2167-2192) amplified the conserved sequences of the VPI capsid region (Robertson et al., 1989), and a probe (22332252) was used to confirm the specificity. For SRSV detection, four sets of primers were selected. A degenerate oligonucleotide (NVpllO, 4865-4884) which can prime cDNA synthesis from all three serogroups of human caliciviruses was used in RT reaction (Le Guyader et al., 1996a). Subsequent PeR amplification was performed with different upstream primers, NVp36 (4487-4501), NVp69 (4733-4752), NI (4768-4788), SR48-SR50-SR52 (4766-4786), for detection of all the different strains (Wang et al., 1994; Ando et al., 1995; Green et al., 1995). To confirm the specificity of these PCR products, five probes were used: PI16 (4796-4815), PI17 (4793-4812), PI18 (4796-4815) (Le Guyader et al., 1996a) and SR47 (4817. 4836),SR61 (4817-4836) (Ando et al., 1995). For rotavirus detection, the primers selected amplified a portion of the VP7 gene of group A RV, Beg 9 (376-392) and R2 (376-392) (Flores et al., 1990; Gouvea et al., 1990). A consensus probe (NFP5) and four probes corresponding to the four major serotypes of group A rotavirus were used (Gouvea et al., 1990; Sethabutr et al., 1990). 2.5~ of the upstream primer in a mix according to the instructions of the murine leukemia virus reverse transcriptase (MuLV-RT) supplier (Perkin Elmer Corp). For dsRV RNA, a previous denaturation step was done by addition of 10mM methylmercuric hydroxide in a mixture containing the sample and the downstream primer. After incubation at room temperature for 5min, other components of the RT mix were added (Dubois et al., 1997). After
RT-PCR - 2J.LL of extracted nucleic acids were used for reverse transcription with
RT-PCRevaluationof viral contamination in shellfishbeds
47
incubation at 42°C for 15min (45min for RV) and denaturation for 5min at 95°C, the tubes were chilled directly on ice. The PCR master mix was added to yield a mixture containing I~M each of the downstream and upstream primers, according to the instructions of the Taq polymerase manufacturer (Perkin-Elmer Corp). Cycling conditions were as follows: initial heat denaturation at 94°C for I min; 40 cycles of denaturation at 94cC for 30s, annealing at 50°C for 30s, extension at noc for 30s and final extension at noc for 7min in a thermocycler (9600 Cycler, Perkin-Elmer Corp). The amplified products were detected by electrophores is on a 9% polyacrylamide gel followed by staining with ethidium bromide. Detection of inhibitor compounds - to monitor for inhibitors, all the samples were co-amplified with ssRNA or dsRNA IC (introduction into the RT mix of the last dilution of IC detectable by RT-PCR). When inhibitory compounds were present, an additional purification step was done: for ssRNA onto a G1SO Sephadex column or for dsRNA by adsorption onto granular cellulose as previously described (Le Guyader et al., 1994). A new extraction was sometimes performed. When no inhibitors were detected, a new round of RT-PCR was performed without IC in order to avoid false negative results due to competition. For EV, as the IC was recognized by the probe, another set of primers (E I and E2) was used to avoid low contamination detectable only by hybridization. The dsIC was not recognized by the probe. Hybridization - for dot blots, PCR product was diluted in a buffer (l0mM trisIHCI pH 8.0; hnM EDTA pH 8.0), denatured for 5min at 95 cC and chilled directly on ice. PCR products were blotted onto a positivelycharged nylon membrane under vacuum and fixed for 5 min by UV crosslinking, as previously described. All probes were labeled with digoxigenin using the 3' tailing kit (Boehringer Mannheim). After prehybridization for 30min at 50°C, hybridization was performed at 50°C for 2h (except for RV probes the temperature was 42°C). The hybridized probes were detected according to the manufacturer's protocol by chemiluminescence (Boehringer Mannheim). Bacteriological analysis tissue and liquor were homogenized in a Warring blender with I volume of 10% (w/v) NaCI-water. FC were determined by conductance measurement (Dupont et al., 1996) and Salmonella by the technique described by the Circulaire DGAUSVHNC81N0. 8003 du 28 avril 1988. »
RESULTS After amplification and hybridization, 34 samples were negative (32.7%) and 69 samples were positive for at least one virus (66%) (Table 1). Sites C-E, occasionally contaminated by FC, showed a lower viral contamination than sites A-B (42.8% negative versus 17%). Most of the samples (68%) from points A-B were contaminated by at least two, three or four different types of virus (36.5%, 19.5% and 12% respectively). HAV were detected only in samples from these two sites and always with other viruses. Table I. Number of negative and positive samples VIlUS
detected
ovirus I virus 2 viruses 3 viruses 4 viruses
Number (%) positive samples Total PointsA-B PointsC-E (n> 104) (0 = 41) (n = 63) 34 (32.7) 7 (17.0) 27 (42.8) 26 (25.0) 6 (14.6) 20 (31.7) 25 (24.0) IS (36.5) to (15.8) 8 (19.5) 14 (13.4) 6 (9.6) 5 (12.0) 4 (4.8) o
Most of the negative samples (87%) showed bacterial contamination of less than 300 FC (Table 2) and 54.5% of samples contaminated by at least three types of viruses had an FC concentration <300 MPNlloog and 45.4% >300 MPN/loog. Three of the positive samples contaminated by all viral types screened (BV, SRSV, RV and HAV) were also highly contaminated by FC and Salmonella . One sample contaminated by the four viral types had a low concentration of FC (48 MPNltoog) (Table 2).
F. LE GUYADER et at.
48
Table 2. Correlation between viral and bacterial contamination Virus detected
o virus
<300 27 20 14
I virus 2 viruses 3 viruses 6 4 viruses I ·SaI = Salmonella contamination.
Faecal coliform concentration >300 >300 + Sal· 4 I 3 0 6 I 5 I
4
3
Total Not known 3 3
5 3
o
31 23 20 II 4
EV. SRSV and RV were detected respectively in 44%, 36.5% and 46% of the shellfish samples analyzed (Table 3) and twice as frequently at the two sites known to be contaminated by FC as at the three sites with occasional bacterial contamination. Table 3. Occurrence of viruses Virus detected
HAV SRSV RV EV
Number (%) positive for each virus SitesC-E SitesA-B Total (n = 63) (n = 104) (n = 41) 6 (14.6) o 6 (5.7) 21 (51.0) 17 (27 .0) 38 (36.5) 48 (46.0) 21 (33.0) 27 (65.8) 26 (63.4) 46 (44.0) 20 (31.7)
RV and EV occurred most frequently but were rarely detected together when samples were contaminated by only two viruses . Each was detected as a single contamination in nine samples. whereas SRSV was detected alone in only three samples and HAV in none. SRSV was associated with EV in four samples and with RV in nine samples. DISCUSSION A variety of methods have been used to recover virus from shellfish . For example. Jaykus et al. (1996) proposed a method that required two days, based on the isolation of intact virions. that enabled direct comparison of detection by RT-PCR and cell culture infectivity . Lopez-Sabater et al. (1997) developed a magnetic immunoseparation PCR assay for HAV detection in raw oysters. A good sensitivity for HAV detection was achieved using a total RNA extraction from oyster meat (Cromeans et al., 1997). Atmar et al. (1995) developed a method for the detection of Norwalk virus and hepatitis A virus from shellfish tissues. Some advantages of this method such as specificity, reliability and reproducibility have been demonstrated (Atmar et al., 1996; Le Guyader et al., 1997). The results reported here showed that the method has proved suitable for analysis of shellfish samples from natural beds. The sensitivity threshold was sufficient to detect positive results after gel electrophoresis (data not shown). and the specificity of the amplification was always confirmed by the hybridization step. which enhanced the sensitivity of detecting virus in some samples with a lower level of contamination. The use of IC gave more reliable results since it detected false-negative samples (mon itoring of inhibitors) and helped prevent false-positive samples (IC avoided the use of a positive control source for contamination). We will develop the use of these IC for the quantitative determination of the level of the viral contamination. This method was originally developed for Norwalk virus and HAV; however, our results showed that RV and EV could also be detected. This study was the first to analyse viral contamination of shellfish beds by RT-PCR over a 2 l-month period. While our results are useful, the time period monitored proved too short for determinations about the seasonality of contamination or demonstration of a clear relationship between the different viruses screened and faecal indicators. The results obtained during this study allow for some conclusions and comments. About 66% of the samples were contaminated. Discussion about this result is difficult since very few data
RT-PCR evaluation of viral contamination in shellfish beds
49
are available in the literature and conditions are always different (methods, sites, samples). In a previous study using a method based on whole shellfish analysis and RT-PCR, we detected EV, RV or HAV in about 40% of samples (Le Guyader et al., 1994). However, in the present study RV or EV occurred more frequently (about 30%) than HAV (20%). The higher frequency of HAV in the earlier study may have been due to a hepatitis A outbreak six months before. Lees et al. (1995) (using a method based on whole shellfish, glass powder matrix and guanidium isothiocyaniate extraction before RT-PCR) detected 21% samples positive for SRSV in shellfish collected at a highly polluted field site and in 8% of samples in a commercial growing area. In 1980. Sobsey et al. (1980) reported infectious enterovirus detection only in oyster samples collected in non-approved areas but with no correlation with the presence of Salmonella. Vaughn et al. (1980) isolated enteroviruses from approved area oyster samples (25%) and closed area samples (37.5%). Goyal et al. (1979) reported the detection of enteric viruses in oyster samples meeting the current bacteriological standards for shellfish harvesting. Our results showed that the two sites regularly contaminated by FC had the highest percentage of viral contamination and HAV was detected only at these sites. These results confirm the advantage of using FC as a standard for legislative control since viruses are more frequently detected in highly polluted areas. However. although the probability of detecting viruses in samples with high FC contamination seems greater. sampling sites meeting the criteria for commercialization presented occasional viral contamination. These results raise the question of consumer risk. Quantification of the level of viral contamination and an epidemiologic survey of outbreaks could help answer this. Molecular epidemiology is needed to compare strains detected in the environment and circulating among the population. ACKNOWLEDGEMENTS This work was supported by a grant from the Environment and Health ministries. We are grateful to JC Sauvagnargues and collaborators (IFREMER. Setes) for sample collection and bacteriological analysis. We thank MK Estes (Baylor College of Medicine, Houston, Texas) for critical review of the manuscript. REFERENCES Ando, T.• Monroe. S. S.• Gentsch, J. R.• Jin, Q. Lew is. D. C. and Glass. R. I. (1995). Detection and differentiation of anti genically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and Southern hybrid ization . J. CUn. Microbiol.• 33, 64-71. Atmar, R. L.. Neill. F. H.• Romalde, J. L.• Le Guyader, F.• Woodley. C. M.• Metcalf. T . G. and Estes, M. K. (1995). Detection of Norwalk virus and Hepatitis A virus in shellfish tissues using the polymerase chain reaction. Appl. Environ. Microbiol., 61.3014-3018. Atmar, R. L.. Neill, F. H.• Woodley, C. M., Manger. R., Fout, G. S., Burkhardt. W., Leja, L.. McGoeven, E. R.. Le Guyader, F., Metcalf, T. G. and Estes . M. K. (1996). Collaborative evaluation of a method for the detection of Norwalk virus in shellfish tissues by PCR. Appl. Environ. Microbiol.• 61. 254-258. Chapman . N. M.• Tracy . S.• Gauntt. C. J. and Fortmueller, U. (1990). Molecular detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybridization. J. Clin. MicrobioL . 28, 834-850. Cremeans, T. L. Nainan, O. V. and Margolis, H. S. (1997). Detection of hepatitis A virus RNA in oyster meat. Appl. Environ. Microbiol.• 63. 2460-2463. Dubois. E.• Le Guyader, F.• Haugarreau, L.• Kopecka, H.• Cormier. M. and Pommepuy, M. (1997) . Molecular epidemiology survey of rotav iruses in sewage by reverse transcriptase seminested PCR and restriction length polymorphism assay . Appl. Environ. Microbiol. , 63. 1794-1800. Dubois. E.• Le Guyader, F.• Kopecka, H. and Pornmepuy, M. (submitted). Analysis of simian rotavirus stability in seawater by cell cul ture and competitive reverse transcriptase-PCR . Dupont. 1.. Menard. D., Herve, C.• Chevcallier, F.• Beliaeff, B. and Minier, B. (1996). Rapid estimation of Escherichia coli in live marine bivalve shellfish using automated conductance measurement. J. Appl. Bact.• 80, 81-90. Flores, 1., Sears. 1.. Perez Schael, L., White. L.• Garcia. D.• Lanata, C. and Kapikian, A. Z. (1990) . Identification of human rotavirus serotype by hybridization to polymerase chain reaction generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7. J. Viral.• 64, 4021·4024. Goyal . S. M.• Gerba, C. P. and Melnick, J. L. (1979) . Human enteroviruses in oysters and their overlying waters. AppL Environ. Microbiol. , 37. 572-581 . Green. 1.. Gallimore. C. I., Norcott, 1. P., Lewis , D. and Brown, D. W. G. (1995). Broadly reactive reverse transcriptase polymerase chain reaction (RT-PCR) for the diagnosis of SRSV -associated gastroenteritis. J. Med. Virol., 47, 392-398 .
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Gouvea, V., Glass . R. I., Woods . P.. Taniguchi, K., Clark . H. F., Forrester. B. and Fang. Z. Y. (1990). Polymerase chain reaction amplification and typing of rotav irus nucleic acid from stool specimens. J. Clin. Microbial.. 28. 276-282. Jaykus, L. A.• De Leon, R. and Sobsey, M. D. (1996) . A virion concentration method for detection of human enteric viruses in oysters by PCR and oligoprobe hybridization. Appl. Environ. Microbial.• 62, 2074-2080. Kopecka, H., Dubrou, S., Prevot, J., Marechal , J. and Lopez-Pila, J. M. (1993). Detection of naturally occurring enteroviruses in water by reverse transcription, polymerase chain reaction and hybridization. Appl. Environ. Microbial.• 59, 1213-1219. Lees. D. N., Henshilwood, K.• Green. J., Gallimore, C. I., Brown, D. W. G. (1995) . Detection of small round structured viruses in shellfish by reverse transcription-PCR. Appl. Environ. Mtcrobtol., 61,4418-4424. Le Guyader, F.• Dubois, E., Menard. D. and Pommepuy, M. (1994). Detection of hepatitis A. rotavirus and enterovirus in naturally contaminated shellfish and sediment by reverse transcription-seminested PCR. Appl. Environ: Microbiol., 60, 3665-3671. Le Guyader, F.• Estes , M. K., Hardy. M. E.• Neill, F. H.• Green. J•• Brown, D. and Atmar, R. L. (I 996a) . Evaluation of a degenerate primer for the detection of human calciv iruses. Arch. ViraL. 141. 2225-2235 . Le Guyader, F.• Neill, F. H., Estes. M. K., Monroe, S. S.• Ando, T. and Atmar, R. L. (l996b). Detection and analysis of an SRSV strain in oysters implicated in an outbreak. Appl. Environ. Microbial., 62, 4268-4272. Le Guyader, F.• Menard. D.• Dubois, E.• Haugarreau, L.• Kopecka, H. and Pommepuy, M. (1997) Use of an internal control to evaluate viral depuration by RT·PCR. IVaI. Sci. Tech., 35,461 -465. Lopez-Sabaier, E. J.. Deng, M. Y. and Cliver. D. O. (1997) . Magnetic immunoseparation PCR assay (MIPA) for detection of ~ hepatit is A virus (HA V) in american oyster (Crassostrea virginica). Lett. Appl. Mlcrobiol., 24, 101-104 . Metcalf, T. G., Melnick, J. L. and Estes, M. K. (1995). Environmental virology : from detection of virus in sewage and water by isolation to identification by molecular biology- a trip of over 50 years . Ann. Rev. Microbial.. 49, 461-487 . Rippey, S. R. (1994) . Infectious diseases associated with molluscan shellfish consumption. Clin. Microbial. Rev., 7, 419-425. Robertson, B. H.• Brown, V. K. and Khanna, B. (1989) . Altered hepatitis A VPI protein resulting from cell culture propagation of virus . Virus Res., 13,207-212. Rotbart, H. A. (1990) . Enzymatic RNA amplification of the enteroviruses. J. Clin. Microbiol., 2.8. 438-442. Sethabutr, 0 .• Uniccmb, L. E., Holmes . I. H.• Taylor. D. N.• Bishop, R. F. and Echeverna, P. (1989) . Serotyping of human group A rotav irus with oligonucleotide probes . J. lnf: Dis.• 162, 368-372 . Sobsey, M. D., Hackney. C. R., Carrick, R. J.. Ray, B. and Speck, M. L. (1980). Occurrence of enteric bacteria and viruses in oysters. J. Food. Prot., 43, 111-113. Vaughn, M., Landry, E. F., Vicale, T. J. and Dahl, M. C . (1980) . Isolation of naturally occurring enteroviruses from a variety of shellfish species resid ing in long Island and New Jersey marine embayments, J. Food. Prot.• 43. 95-98. Wang, J., Jiang , X.• Madore, H. P., Gray , I .. Desselberger, U.. Ando, T.• Seto, Y.• Oishi. I., Lew, J. P., Green . K. Y. and Estes. M. K. ( 1994). Sequence divers ity of small, round-structured viruses in the Norwalk virus group . J. Yirol.• 68, 5982-5990.
Pergamon
War. ScL
PIT: S0273-1223(98)00798-7
T~ch .
Vol. 38. No. 12. pp. 4S-S0. 1998. e 1998IAWQ Published by Elsevier Science lid
Printed in GreatBritain. All rightsreserved 0273-1223/98 $ 19-00 + 0 '00
RT-PCR EVALUAnON OF VIRAL CONTAMINATION IN FIVE SHELLFISH BEDS OVER A 21-MONTH PERIOD F. Le Guyader*, L. Miossec*, L. Haugarreau*. E. Dubois*, H. Kopecka** and M. Pommepuy* • Laboratoire de Microbiologie, IFREMER. BP 21105.44311 Nantescedex03. France •• Unitede YirologieMoliculaire. lnstitut Pasteur. 28 rue du Dr Raux; 75/24 Pariscedex /5. France
ABSTRACf Five shellfish beds were sampled for 21 months and evaluated for microbial contamination . Viral extraction was performed on dissected tissues and the clinically most important enteric viruses (hepatitis A virus. small round structured virus. rotavirus and enterovirus) were searched for by RT·PCR and hybridization. Among the 104 samples analysed. 66% were contaminated by at least one virus and 34% were negative for any virus. The two sites regularly contaminated by faecal coliforms had the highest percentage of viral contamination and HAV was detected only in these sites. However. sampling sites meeting the criteria for commercialisation showed occasional viral contamination and viruses were detected in samples with no faecal coliform contamination . e 1998 fAWQ Published by Elsevier Science Ltd. All rights reserved
KEYWORDS Enterovirus; faecal colifomns; hepatitis A virus; rotavirus; shellfish; small round structured virus. INTRODUCfION Shellfish are known to concentrate microorganisms such as bacteria or viruses in association with human waste or bacterial pathogens indigenous to coastal marine environments (Rippey, 1994). It is known that standards based on faecal coliforms (FC) and established to protect shellfish consumers are not correlated with the presence of viruses. In recent years, different methods have been developed to detect viral contamination in shellfish (Atmar et al., 1995; Lees et al., 1995; Cromeans et al., 1997). The latest developments in shellfish viral bio-accumulation and current knowledge about viruses at the molecular level have led to the elaboration of a specific, sensitive method which was evaluated in a COllaborative study with artificially contaminated shellfish (Atmar et al.• 1995, 1996), and used to analyse shellfish implicated in an outbreak (Le Guyader et 01.• 1996b). This paper describes the use of this method in assessing the viral Contamination of five different shellfish beds over a 21-month period. The clinically most important viruses likely to be accumulated by shellfish were evaluated by RT-PCR and hybridization: hepatitis A virus (HAV), small round structured virus (SRSV), rotaviruses (RV) and enteroviruses (BV).
45
46
F. LE GUYADER et al.
MATERIALS AND METHODS
Shellfish sampling andJ'rocessing - oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis) were collected monthly from August 1995 to April 1997 at five different sites in southern France known to have different levels of bacterial contamination. Two mussel beds (A-B) were frequently contaminated by faecal coliforms and Salmonella and three oyster beds (C-E) were occasionally contaminated by FC. A total of 42 mussel samples and 63 oyster samples were collected. Shellfish received by the laboratory two days after collection were shucked and the stomach and digestive diverticula removed by dissection. Dissected tissues were frozen (-20°C) in aliquots of 1.5g until processing. For analysis, the tissues were thawed on ice and processed as described previously (Atmar et al., 1995). Briefly, the stomach and digestive diverticula were homogenized with a Potter-Elvehjem homogenizer (Bioblock scientific, Illkirch) in 2mL of phosphatebuffered saline solution (PBS, pH 7.4). The potter was rinsed with an additional 3mL of PBS which was then added to the homogenate. After addition of one volume of chloroform-butanol (I: I v/v), the mix was vortexed for Imin and 90J.LL of Cat-Floc (Calgon Corp, Elwood, Pa) were added. After rocking for 5min and settling for 15min, the mixture was centrifuged at lO,oooxg for 20min, rocked and centrifuged. The aqueous phase was then added to 3mL of polyethylene glycol 6,000 (Sigma, 24% (w/v)-sodium chloride (1.2M) solution). After rocking for lh at 4°C, the solution was centrifuged for 30min at lO,oooxg. The pellet was then suspended in 3mL of sterile water and digested with proteinase K (Sigma) for 30min at 56°C. The nucleic acids were extracted with an equal volume of phenol-chloroform-isoamyl alcohol (25:24: I) (Sigma) and precipitated with ethanol. After the resulting pellet was suspended in water, cetyltrimethylarnrnonium bromide (CTAB) (1.4% w/v) and sodium chloride (Sigma, O.IIM) were added. The mixture was incubated for 15min at room temperature before being centrifuged for 30min at 15,oooxg at 25°C. The resulting pellet was suspended in IM sodium chloride and precipitated in ethanol before suspension in 50J.LL of water prior to reverse transcription.
Intemal control - to evaluate the presence of inhibitory compounds in the enzymatic reactions, internal controls (IC) were used in RT-PCR. A single-strand RNA IC (ssRNA IC) was constructed from the enterovirus genome (Le Guyader et al., 1997) and a double strand RNA IC (dsRNA IC) was constructed from the rotavirus genome (Dubois et al., submitted). Both IC were quantitated by RT-PCR and by spectroscopy at a wavelength of 260nm before use in RT-PCR. Primers - for enterovirus detection two sets of primers both located in the 5' untranslated region were used. One set, KI (162-182) and K2 (577-596) (Kopecka et al., 1993), amplified both endogenous RNA and the ssRNA IC, whereas the second set, EI (446-463) and E2 (623-642) (Chapman et al., 1990; Rotbart, 1990), amplified only endogenous RNA. A specific oligonucleotide probe (533-559) (Chapman et al., 1990) was used to hybridize this fragment. For hepatitis A virus, primers HI (2389-2413) and H2 (2167-2192) amplified the conserved sequences of the VPI capsid region (Robertson et al., 1989), and a probe (22332252) was used to confirm the specificity. For SRSV detection, four sets of primers were selected. A degenerate oligonucleotide (NVpllO, 4865-4884) which can prime cDNA synthesis from all three serogroups of human caliciviruses was used in RT reaction (Le Guyader et al., 1996a). Subsequent PeR amplification was performed with different upstream primers, NVp36 (4487-4501), NVp69 (4733-4752), NI (4768-4788), SR48-SR50-SR52 (4766-4786), for detection of all the different strains (Wang et al., 1994; Ando et al., 1995; Green et al., 1995). To confirm the specificity of these PCR products, five probes were used: PI16 (4796-4815), PI17 (4793-4812), PI18 (4796-4815) (Le Guyader et al., 1996a) and SR47 (4817. 4836),SR61 (4817-4836) (Ando et al., 1995). For rotavirus detection, the primers selected amplified a portion of the VP7 gene of group A RV, Beg 9 (376-392) and R2 (376-392) (Flores et al., 1990; Gouvea et al., 1990). A consensus probe (NFP5) and four probes corresponding to the four major serotypes of group A rotavirus were used (Gouvea et al., 1990; Sethabutr et al., 1990). 2.5~ of the upstream primer in a mix according to the instructions of the murine leukemia virus reverse transcriptase (MuLV-RT) supplier (Perkin Elmer Corp). For dsRV RNA, a previous denaturation step was done by addition of 10mM methylmercuric hydroxide in a mixture containing the sample and the downstream primer. After incubation at room temperature for 5min, other components of the RT mix were added (Dubois et al., 1997). After
RT-PCR - 2J.LL of extracted nucleic acids were used for reverse transcription with
RT-PCRevaluationof viral contamination in shellfishbeds
47
incubation at 42°C for 15min (45min for RV) and denaturation for 5min at 95°C, the tubes were chilled directly on ice. The PCR master mix was added to yield a mixture containing I~M each of the downstream and upstream primers, according to the instructions of the Taq polymerase manufacturer (Perkin-Elmer Corp). Cycling conditions were as follows: initial heat denaturation at 94°C for I min; 40 cycles of denaturation at 94cC for 30s, annealing at 50°C for 30s, extension at noc for 30s and final extension at noc for 7min in a thermocycler (9600 Cycler, Perkin-Elmer Corp). The amplified products were detected by electrophores is on a 9% polyacrylamide gel followed by staining with ethidium bromide. Detection of inhibitor compounds - to monitor for inhibitors, all the samples were co-amplified with ssRNA or dsRNA IC (introduction into the RT mix of the last dilution of IC detectable by RT-PCR). When inhibitory compounds were present, an additional purification step was done: for ssRNA onto a G1SO Sephadex column or for dsRNA by adsorption onto granular cellulose as previously described (Le Guyader et al., 1994). A new extraction was sometimes performed. When no inhibitors were detected, a new round of RT-PCR was performed without IC in order to avoid false negative results due to competition. For EV, as the IC was recognized by the probe, another set of primers (E I and E2) was used to avoid low contamination detectable only by hybridization. The dsIC was not recognized by the probe. Hybridization - for dot blots, PCR product was diluted in a buffer (l0mM trisIHCI pH 8.0; hnM EDTA pH 8.0), denatured for 5min at 95 cC and chilled directly on ice. PCR products were blotted onto a positivelycharged nylon membrane under vacuum and fixed for 5 min by UV crosslinking, as previously described. All probes were labeled with digoxigenin using the 3' tailing kit (Boehringer Mannheim). After prehybridization for 30min at 50°C, hybridization was performed at 50°C for 2h (except for RV probes the temperature was 42°C). The hybridized probes were detected according to the manufacturer's protocol by chemiluminescence (Boehringer Mannheim). Bacteriological analysis tissue and liquor were homogenized in a Warring blender with I volume of 10% (w/v) NaCI-water. FC were determined by conductance measurement (Dupont et al., 1996) and Salmonella by the technique described by the Circulaire DGAUSVHNC81N0. 8003 du 28 avril 1988. »
RESULTS After amplification and hybridization, 34 samples were negative (32.7%) and 69 samples were positive for at least one virus (66%) (Table 1). Sites C-E, occasionally contaminated by FC, showed a lower viral contamination than sites A-B (42.8% negative versus 17%). Most of the samples (68%) from points A-B were contaminated by at least two, three or four different types of virus (36.5%, 19.5% and 12% respectively). HAV were detected only in samples from these two sites and always with other viruses. Table I. Number of negative and positive samples VIlUS
detected
ovirus I virus 2 viruses 3 viruses 4 viruses
Number (%) positive samples Total PointsA-B PointsC-E (n> 104) (0 = 41) (n = 63) 34 (32.7) 7 (17.0) 27 (42.8) 26 (25.0) 6 (14.6) 20 (31.7) 25 (24.0) IS (36.5) to (15.8) 8 (19.5) 14 (13.4) 6 (9.6) 5 (12.0) 4 (4.8) o
Most of the negative samples (87%) showed bacterial contamination of less than 300 FC (Table 2) and 54.5% of samples contaminated by at least three types of viruses had an FC concentration <300 MPNlloog and 45.4% >300 MPN/loog. Three of the positive samples contaminated by all viral types screened (BV, SRSV, RV and HAV) were also highly contaminated by FC and Salmonella . One sample contaminated by the four viral types had a low concentration of FC (48 MPNltoog) (Table 2).
F. LE GUYADER et at.
48
Table 2. Correlation between viral and bacterial contamination Virus detected
o virus
<300 27 20 14
I virus 2 viruses 3 viruses 6 4 viruses I ·SaI = Salmonella contamination.
Faecal coliform concentration >300 >300 + Sal· 4 I 3 0 6 I 5 I
4
3
Total Not known 3 3
5 3
o
31 23 20 II 4
EV. SRSV and RV were detected respectively in 44%, 36.5% and 46% of the shellfish samples analyzed (Table 3) and twice as frequently at the two sites known to be contaminated by FC as at the three sites with occasional bacterial contamination. Table 3. Occurrence of viruses Virus detected
HAV SRSV RV EV
Number (%) positive for each virus SitesC-E SitesA-B Total (n = 63) (n = 104) (n = 41) 6 (14.6) o 6 (5.7) 21 (51.0) 17 (27 .0) 38 (36.5) 48 (46.0) 21 (33.0) 27 (65.8) 26 (63.4) 46 (44.0) 20 (31.7)
RV and EV occurred most frequently but were rarely detected together when samples were contaminated by only two viruses . Each was detected as a single contamination in nine samples. whereas SRSV was detected alone in only three samples and HAV in none. SRSV was associated with EV in four samples and with RV in nine samples. DISCUSSION A variety of methods have been used to recover virus from shellfish . For example. Jaykus et al. (1996) proposed a method that required two days, based on the isolation of intact virions. that enabled direct comparison of detection by RT-PCR and cell culture infectivity . Lopez-Sabater et al. (1997) developed a magnetic immunoseparation PCR assay for HAV detection in raw oysters. A good sensitivity for HAV detection was achieved using a total RNA extraction from oyster meat (Cromeans et al., 1997). Atmar et al. (1995) developed a method for the detection of Norwalk virus and hepatitis A virus from shellfish tissues. Some advantages of this method such as specificity, reliability and reproducibility have been demonstrated (Atmar et al., 1996; Le Guyader et al., 1997). The results reported here showed that the method has proved suitable for analysis of shellfish samples from natural beds. The sensitivity threshold was sufficient to detect positive results after gel electrophoresis (data not shown). and the specificity of the amplification was always confirmed by the hybridization step. which enhanced the sensitivity of detecting virus in some samples with a lower level of contamination. The use of IC gave more reliable results since it detected false-negative samples (mon itoring of inhibitors) and helped prevent false-positive samples (IC avoided the use of a positive control source for contamination). We will develop the use of these IC for the quantitative determination of the level of the viral contamination. This method was originally developed for Norwalk virus and HAV; however, our results showed that RV and EV could also be detected. This study was the first to analyse viral contamination of shellfish beds by RT-PCR over a 2 l-month period. While our results are useful, the time period monitored proved too short for determinations about the seasonality of contamination or demonstration of a clear relationship between the different viruses screened and faecal indicators. The results obtained during this study allow for some conclusions and comments. About 66% of the samples were contaminated. Discussion about this result is difficult since very few data
RT-PCR evaluation of viral contamination in shellfish beds
49
are available in the literature and conditions are always different (methods, sites, samples). In a previous study using a method based on whole shellfish analysis and RT-PCR, we detected EV, RV or HAV in about 40% of samples (Le Guyader et al., 1994). However, in the present study RV or EV occurred more frequently (about 30%) than HAV (20%). The higher frequency of HAV in the earlier study may have been due to a hepatitis A outbreak six months before. Lees et al. (1995) (using a method based on whole shellfish, glass powder matrix and guanidium isothiocyaniate extraction before RT-PCR) detected 21% samples positive for SRSV in shellfish collected at a highly polluted field site and in 8% of samples in a commercial growing area. In 1980. Sobsey et al. (1980) reported infectious enterovirus detection only in oyster samples collected in non-approved areas but with no correlation with the presence of Salmonella. Vaughn et al. (1980) isolated enteroviruses from approved area oyster samples (25%) and closed area samples (37.5%). Goyal et al. (1979) reported the detection of enteric viruses in oyster samples meeting the current bacteriological standards for shellfish harvesting. Our results showed that the two sites regularly contaminated by FC had the highest percentage of viral contamination and HAV was detected only at these sites. These results confirm the advantage of using FC as a standard for legislative control since viruses are more frequently detected in highly polluted areas. However. although the probability of detecting viruses in samples with high FC contamination seems greater. sampling sites meeting the criteria for commercialization presented occasional viral contamination. These results raise the question of consumer risk. Quantification of the level of viral contamination and an epidemiologic survey of outbreaks could help answer this. Molecular epidemiology is needed to compare strains detected in the environment and circulating among the population. ACKNOWLEDGEMENTS This work was supported by a grant from the Environment and Health ministries. We are grateful to JC Sauvagnargues and collaborators (IFREMER. Setes) for sample collection and bacteriological analysis. We thank MK Estes (Baylor College of Medicine, Houston, Texas) for critical review of the manuscript. REFERENCES Ando, T.• Monroe. S. S.• Gentsch, J. R.• Jin, Q. Lew is. D. C. and Glass. R. I. (1995). Detection and differentiation of anti genically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and Southern hybrid ization . J. CUn. Microbiol.• 33, 64-71. Atmar, R. L.. Neill. F. H.• Romalde, J. L.• Le Guyader, F.• Woodley. C. M.• Metcalf. T . G. and Estes, M. K. (1995). Detection of Norwalk virus and Hepatitis A virus in shellfish tissues using the polymerase chain reaction. Appl. Environ. Microbiol., 61.3014-3018. Atmar, R. L.. Neill, F. H.• Woodley, C. M., Manger. R., Fout, G. S., Burkhardt. W., Leja, L.. McGoeven, E. R.. Le Guyader, F., Metcalf, T. G. and Estes . M. K. (1996). Collaborative evaluation of a method for the detection of Norwalk virus in shellfish tissues by PCR. Appl. Environ. Microbiol.• 61. 254-258. Chapman . N. M.• Tracy . S.• Gauntt. C. J. and Fortmueller, U. (1990). Molecular detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybridization. J. Clin. MicrobioL . 28, 834-850. Cremeans, T. L. Nainan, O. V. and Margolis, H. S. (1997). Detection of hepatitis A virus RNA in oyster meat. Appl. Environ. Microbiol.• 63. 2460-2463. Dubois. E.• Le Guyader, F.• Haugarreau, L.• Kopecka, H.• Cormier. M. and Pommepuy, M. (1997) . Molecular epidemiology survey of rotav iruses in sewage by reverse transcriptase seminested PCR and restriction length polymorphism assay . Appl. Environ. Microbiol. , 63. 1794-1800. Dubois. E.• Le Guyader, F.• Kopecka, H. and Pornmepuy, M. (submitted). Analysis of simian rotavirus stability in seawater by cell cul ture and competitive reverse transcriptase-PCR . Dupont. 1.. Menard. D., Herve, C.• Chevcallier, F.• Beliaeff, B. and Minier, B. (1996). Rapid estimation of Escherichia coli in live marine bivalve shellfish using automated conductance measurement. J. Appl. Bact.• 80, 81-90. Flores, 1., Sears. 1.. Perez Schael, L., White. L.• Garcia. D.• Lanata, C. and Kapikian, A. Z. (1990) . Identification of human rotavirus serotype by hybridization to polymerase chain reaction generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7. J. Viral.• 64, 4021·4024. Goyal . S. M.• Gerba, C. P. and Melnick, J. L. (1979) . Human enteroviruses in oysters and their overlying waters. AppL Environ. Microbiol. , 37. 572-581 . Green. 1.. Gallimore. C. I., Norcott, 1. P., Lewis , D. and Brown, D. W. G. (1995). Broadly reactive reverse transcriptase polymerase chain reaction (RT-PCR) for the diagnosis of SRSV -associated gastroenteritis. J. Med. Virol., 47, 392-398 .
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